Long-term presence of autoantibodies in plasma of cured leprosy patients

Autoantibodies have been detected in leprosy patients, indicating that infection with M. leprae may lead to autoimmune disorders. However, whether autoimmune response last until patients are cured is unknown. Knowing the autoimmune response in cured leprosy patients is essential to identify whether symptoms are caused by leprosy itself or by other immune-related diseases. This knowledge is essential for the ongoing health management in cured leprosy patients where autoimmune disorders still exist. In our study, we selected six autoantibodies, including anticardiolipin antibody of IgG (ACA), anti-nuclear antibody (ANA), extractable nuclear antigen antibody (ENA), anti-streptolysin O (ASO), anti-double stranded DNA antibody (dsDNA), and rheumatoid factor (RF), that had been reported in leprosy patients as typical autoantibodies. We tested the six typical autoantibodies combined with LACC1, which encodes a protein associated with autoimmune disease such as Crohn’s disease and is also the susceptible gene conferring leprosy risk, in cured leprosy patients through ELISA to assess the cured patient’s immune status. We observed high positive rates of autoantibodies in cured leprosy patients, and the average plasma levels of five (ACA, ANA, ENA, ASO, and RF) out of the six autoantibodies were significantly higher in cured leprosy patients than in controls. The positive detection of autoantibodies is independent of the recovery period. Moreover, the level of these autoantibodies showed a strong positive correlation with the level of LACC1 in both controls and cured patients. This study showed that there is long-term autoimmunological activation in leprosy patients, even after decades of recovery. Autoimmune responses may influence the development and prognosis of leprosy. Special care should be given to posttreatment or cured leprosy patients regarding long-term autoimmunological activation.

High positive rate of plasma autoantibodies in cured leprosy patients. We measured the levels of ACA-IgG, ENA-Ab, ASO-Ab, RF-IgA, ANA-Ab, dsDNA-Ab, and LACC1-Ab in the plasma of patients and controls. Since normal individuals have low levels of autoantibodies, an individual with a plasma level of an autoantibody higher than the mean + 2 SD (standard deviation) level of that of the control group was defined as positive for the autoantibody, as based on the manufacturer's instructions for the ELISA kit. The concentration of ANA > 87.3 ng/mL, ACA-IgG > 43.6 ng/mL, dsDNA > 16.7 ng/mL, ASO > 329.9 U/L, ENA-Ab > 87.4 ng/mL, LACC1 > 3.7 ng/mL, RF > 3.0 ng/mL were thus defined as positive, respectively. As such, higher positive rates of all six detected autoantibodies were observed in cured leprosy patients: 91 (46.0%), 30 (15.2%), 74 (37.4%), 55 (27.8%), 95 (48.0%), and 26 (13.1%) patients were positive for ACA, ENA, ASO, ANA, RF, and dsDNA, respectively; while the positive rate of these autoantibodies ranges around 3-5% in normal controls. There were no significant differences in LACC1, ACA, ENA, ASO, ANA, dsDNA and RF concentrations between different leprosy subtypes (P > 0.05). Intriguingly, in the BB subtype, we observed the highest positive rate of ACA but the lowest positive rates of ENA, ASO and ANA (Table 2). In contrast to previous findings in the course of the disease showing that ACA-IgG was significantly higher in lepromatous leprosy and multibacillary patient subgroups 5 , no extremely higher positive rate was observed for lepromatous leprosy in our study. Notably, the positive rates of ASO (45.8%) and RF (61.0%) were extremely high in the BL subtype.
Long-term presence of plasma autoantibodies in cured leprosy patients. In addition to the positive rate, the average levels of these autoantibodies, ACA, ANA, ENA, ASO, and RF, were significantly higher in Table 1. Demographic and clinical characteristics of individuals enrolled in this study.  www.nature.com/scientificreports/ the plasma of the patient group than in that of the controls (Fig. 1A). Sex had no effect on the plasma levels of these autoantibodies in either controls or patients, except for ACA in controls (Fig. 1B,C). Age had no effect on the plasma levels of these autoantibodies in controls ( Fig. 2A), whereas the plasma levels of all six autoantibodies were positively related to age in patients (Fig. 2B). We also found that there was no statistically significant difference in autoantibody concentrations between different disability levels or treatments. Nevertheless, there were weak correlations between the levels of the autoantibodies and the recovery time period (Fig. 2C): the autoantibodies presented in both subjects in the period of onset or subjects in the period after decades of recovery (P = 0.045, 0.734, 0.071, 0.039, 0.236, 0.661 for ACA, ENA, ASO, ANA, RF and dsDNA respectively). Table 2. Positive rate of ACA-IgG, ENA-Ab, ASO-Ab, RF-IgA, ANA-Ab, dsDNA-Ab, LACC1-Ab in the plasma of cured leprosy patients and controls. a TT, tuberculoid leprosy, BT, borderline tuberculoid leprosy; BB, mid-borderline leprosy; BL, borderline lepromatous leprosy; LL, lepromatous leprosy; b +, positive, an individual with the plasma level of an autoantibody higher than the mean + 2 SD (standard deviation) level of that of the control group was defined as positive for the autoantibody. c Highest rate in the five subtypes were marked in bold and italic. d Comparisons of the positive rate of each autoantibody between control and patients groups were conducted using correct Chi-Squared Test of four-fold table, in five subtypes were conducted using Chi-Squared Test of contingency table. A P value < 0.05 was considered to be statistical significance.  www.nature.com/scientificreports/ Positive correlation between the levels of autoantibodies and the level of the LACC1 protein. As LACC1 is a master regulator in autoimmune diseases, we investigated whether the autoantibody levels were related to LACC1 protein levels. We found that the protein level of LACC1 was similar in cured patients and controls, and the level of LACC1 protein increased slightly with age in patients but remained consistent with ageing in controls (P = 0.443, 0.094 in control group and patient group, respectively) (Fig. 3). Intriguingly, the levels of all six autoantibodies were strongly related to the protein level of LACC1 in both controls and patients (Fig. 2D,E

Discussion
The onset and development of leprosy is dependent on host-pathogen interaction. Since M. leprae could be latent for many years before the clinical manifestations, the autoimmune response might be involved in the course of leprosy. Indeed, we and others have shown that genetic variants within autoimmune-related genes and pathways contribute to the development of leprosy 14 . Clinical observations have shown that M. leprae infection might mimic the performance of systemic lupus erythematosus 4,15 and trigger the reactivation of lupus. Numerous studies have reported autoimmune responses during the course of leprosy 13,16 . However, it is unclear whether the autoimmune response occurs before the onset of leprosy or whether autoimmune activation is just an effect of bacterial infection. We detected the circulating concentrations of six typical autoantibodies in cured leprosy patients with different recovery periods and found that the concentrations of these autoantibodies were significantly higher in cases than in controls. Considering that different methods and materials may affect the results, we defined individuals www.nature.com/scientificreports/ with a plasma level of an autoantibody higher than the mean + 2 SD (standard deviation) level of that of the control group as positive according to the ELISA kit instructions used. The positive rate of these autoantibodies ranges from 13.1 to 48.0%, which was consistent with previously findings in patients with present illness 6,8,[17][18][19] .
Our results showed that age had no effect on the plasma levels of these autoantibodies in controls, whereas in patients the plasma levels of these autoantibodies increase with age, which suggests that the severity of autoimmune disorders caused by M. leprae may be age-related. We also found that there was no statistically significant difference in autoantibody concentrations between different disability levels or treatments. This result indicated that the immune status of cured leprosy patients may not be connected to the severity of disease and drugs. However, the levels of dsDNA and ANA concentrations were higher in the BT group than in the BB group. In the BB subtype, we observed the highest positive rate of ACA but the lowest positive rates of ENA, ASO, ANA, and RF. BB leprosy is an intermediate subtype of leprosy, with a lower bacterial load than LL leprosy and a higher bacterial load than TT leprosy, and the immune response is stronger than LL leprosy and weaker than TT leprosy. The highest and lowest immune antibodies in BB leprosy suggest that the cause of the immune disorder may not be solely due to bacterial or host immunity. Notably, we observed the presence of autoantibodies in both subjects in the period of onset and subjects in the period after decades of recovery, indicating a long-term presence of autoantibodies in cured leprosy patients. Since the cured patients haven't suffered from any autoimmunerelated symptoms, the presence of autoantibodies might be a result of the interaction between M. leprae and the immune system of the host. As our analysis showed that there is no significant correlation between disability and the presence of autoantibodies, what leads to the raise of autoantibodies, and what's the consequence of the autoantibodies, remained to be open questions. This phenomenon might be explained by two possibilities: (1) those subjects suffering from leprosy are more susceptible to autoimmune activation or autoimmune complications, and the presence of autoantibodies in these individuals is the result of genetic predisposition rather than the effect of M. leprae infection. (2) There are continuous immune activations in cured patients due to unknown reasons or potential latent pathogens. For the first possibility, we observed that the levels of all six autoantibodies were strongly related to the protein level of LACC1, which is a key protein in multiple autoimmune-mediated diseases 20 , in both controls and patients, indicating that the genetic predisposition to autoimmunity could partially explain the high positive rate and level of autoantibodies of cured patients. As there were too less relapse and leprosy reaction happened in enrolled patients, we can't effectively analyze whether recurrence and leprosy reaction affected these autoantibodies. Therefore, the presence of autoantibodies decades after recovery might be the result of the long-term effect of M. leprae infection. Additionally, we cannot rule out the possibility that www.nature.com/scientificreports/ dead M. leprae or its debris might trigger the immune system since it is common to find acid-fast bacilli in skin smears even after completion of multidrug therapy.

Conclusions
While the presence of autoantibodies in current leprosy patients might partially explain autoimmune-related complications, the presence of autoantibodies in cured patients indicates a long-term effect of M. leprae infection on the host immune status. Autoimmunological activation might indicate incomplete treatment of the disease or may act as an inducer for subsequent autoimmune conditions. In both cases, special care should be given regarding long-term autoimmunological activation to both posttreatment and cured leprosy patients.

Methods
Demographic information and plasma sample collection.  Sample preparation After thawing, the plasma sample from each individual was centrifuged at 4 °C for 10 min at 3000 R/min and then diluted 4 times with an appropriate dilution buffer and used for the analysis of ACA, ANA,ENA, dsDNA, ASO, RF, and LACC1. The ACA, ANA, ENA, dsDNA, ASO, RF, and LACC1 concentrations were determined by commercial ELISA kits (Jianglai Bio, Shanghai, China) according to the manufacturer's instructions.
Elisa Plasma (1:4 diluted with sample buffer, 50 µL per well) and antibodies (marked by HRP, 100μL per well) were add and then incubated at 37 °C for 1 h. Sample wells were washed five times with a provided wash buffer (20 × dilution with ddH2O, 350μL per well), then the provided substrate solution was added (50 µL per well) and incubated at ambient temperature of 37 °C for 15 min. After a second wash step, the provided stop solution was then added (50 μL per well) and absorbance of sample wells measured immediately at 450 nm in 15 min. Data were then analyzed as recommended by the manufacturer. Statistical analysis Comparisons of the level of each autoantibody in different groups were conducted using the nonparametric Mann-Whitney test. Comparisons of the positive rate of each autoantibody between control and patient groups were conducted using correct Chi-Squared Test of four-fold table, comparisons for five subtypes were conducted using Chi-Squared Test of contingency table. Correlation between the level of autoantibodies and the level of LACC1 protein was measured by Pearson correlation, whereas the alteration of the autoantibodies along age was measured by linear regression. All analyses were carried out with GraphPad Prism (version 8.3.0). P values < 0.05 were considered statistically significant.

Ethics approval and consent participate. The study is approved by the Institutional Review Board of
Kunming Institute of Zoology, Chinese Academy of Sciences, with approval number of SMKX-SQ-20200414-075. All subjects participated had providing written informed consent according to the principles of the Declaration of Helsinki. Subjects under 16 years old were not involved in this study. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.